Low molecular weight cationic lipids for oligonucleotide delivery

ABSTRACT

The instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver. The present invention employs low molecular weight cationic lipids comprising at least one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/880,254 filed Apr. 18, 2013, which is 371 National Phase Entry ofInternational Patent Application No. PCT/US2011/56502 filed on Oct. 17,2011, and which claims benefit under 35 U.S.C. 119(e) of the U.S.Provisional Application No. 61/405,413, filed Oct. 21, 2010, thecontents of which are incorporated herein by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “MRLMIS00046USPCT-SEQTXT-28JUNE2013.txt”, creation date ofJun. 28, 2013 and a size of 3217 bytes. The sequence listing submittedvia EFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to novel cationic lipids that can be usedin combination with other lipid components such as cholesterol andPEG-lipids to form lipid nanoparticles with oligonucleotides, tofacilitate the cellular uptake and endosomal escape, and to knockdowntarget mRNA both in vitro and in vivo.

Cationic lipids and the use of cationic lipids in lipid nanoparticlesfor the delivery of oligonucleotides, in particular siRNA and miRNA,have been previously disclosed. Lipid nanoparticles and use of lipidnanoparticles for the delivery of oligonucleotides, in particular siRNAand miRNA, has been previously disclosed. Oligonucleotides (includingsiRNA and miRNA) and the synthesis of oligonucleotides has beenpreviously disclosed. (See US patent applications: US 2006/0083780, US2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 andPCT patent applications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405,WO2010/054406 and WO2010/105209). See also Semple S. C. et al., Rationaldesign of cationic lipids for siRNA delivery, Nature Biotechnology,published online 17 Jan. 2010; doi:10.1038/nbt.1602.

Other cationic lipids are disclosed in US patent applications: US2009/0263407, US 2009/0285881, US 2010/0055168, US 2010/0055169, US2010/0063135, US 2010/0076055, US 2010/0099738 and US 2010/0104629.

Further, the cationic lipid DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19yl4-(dimethylamino)butanoate) is disclosed in WO2010/105209.

Traditional cationic lipids such as CLinDMA and DLinDMA have beenemployed for siRNA delivery to liver but suffer from non-optimaldelivery efficiency along with liver toxicity at higher doses. It is anobject of the instant invention to provide a cationic lipid scaffoldthat demonstrates enhanced efficacy along with lower liver toxicity as aresult of lower lipid levels in the liver. The present invention employslow molecular weight cationic lipids comprising at least one short lipidchain to enhance the efficiency and tolerability of in vivo delivery ofsiRNA.

SUMMARY OF THE INVENTION

The instant invention provides for novel cationic lipids that can beused in combination with other lipid components such as cholesterol andPEG-lipids to form lipid nanoparticles with oligonucleotides. It is anobject of the instant invention to provide a cationic lipid scaffoldthat demonstrates enhanced efficacy along with lower liver toxicity as aresult of lower lipid levels in the liver. The present invention employslow molecular weight cationic lipids with one short lipid chain toenhance the efficiency and tolerability of in vivo delivery of siRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: LNP (Compound 1) efficacy in mice.

FIG. 2: LNP (Compound 2) efficacy in rats.

FIG. 3: Lipid (Compound 2) levels in rat liver.

DETAILED DESCRIPTION OF THE INVENTION

The various aspects and embodiments of the invention are directed to theutility of novel cationic lipids useful in lipid nanoparticles todeliver oligonucleotides, in particular, siRNA and miRNA, to any targetgene. (See US patent applications: US 2006/0083780, US 2006/0240554, US2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patentapplications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405,WO2010/054406 and WO2010/105209). See also Semple S. C. et al., Rationaldesign of cationic lipids for siRNA delivery, Nature Biotechnology,published online 17 Jan. 2010; doi:10.1038/nbt.1602.

The cationic lipids of the instant invention are useful components in alipid nanoparticle for the delivery of oligonucleotides, specificallysiRNA and miRNA.

In a first embodiment of this invention, the cationic lipids areillustrated by the Formula A:

wherein:

R¹ and R² are independently selected from H, (C₁-C₆)alkyl, heterocyclyl,and polyamine, wherein said alkyl, heterocyclyl and polyamine areoptionally substituted with one to three substituents selected from R′,or R¹ and R² can be taken together with the nitrogen to which they areattached to form a monocyclic heterocycle with 4-7 members optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic heterocycle isoptionally substituted with one to three substituents selected from R′;

R³ is selected from H and (C₁-C₆)alkyl, said alkyl optionallysubstituted with one to three substituents selected from R′;

R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ andCON(R″)₂;

R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkylis optionally substituted with halogen and OH;

n is 0, 1, 2, 3, 4 or 5;

X is selected from O, NR″, (C═O)O, NR″(C═O), O(C═O)O, NR″(C═O)NR″,O(C═O)NR″, and NR″(C═O)O;

L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄ alkenyl, said alkyl andalkenyl are optionally substituted with one or more substituentsselected from R′; and

L₂ is selected from C₃-C₉ alkyl and C₃-C₉ alkenyl, said alkyl andalkenyl are optionally substituted with one or more substituentsselected from R′;

or any pharmaceutically acceptable salt or stereoisomer thereof.

In a second embodiment, the invention features a compound having FormulaA, wherein:

R¹ and R² are each methyl;

R³ is H;

n is 3;

X is (C═O)O;

L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄ alkenyl; and L₂ is selectedfrom C₃-C₉ alkyl and C₃-C₉ alkenyl;

or any pharmaceutically acceptable salt or stereoisomer thereof.

Specific cationic lipids are:

-   (20Z,23Z)-nonacosa-20,23-dien-10-yl 4-(dimethylamino)butanoate    (Compound 1); and-   (18Z)-heptacos-18-en-10-yl 4-(dimethylamino)butanoate (Compound 2);-   or any pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the cationic lipids disclosed are useful in thepreparation of lipid nanoparticles.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of oligonucleotides.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA and miRNA.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA.

The cationic lipids of the present invention may have asymmetriccenters, chiral axes, and chiral planes (as described in: E. L. Elieland S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons,New York, 1994, pages 1119-1190), and occur as racemates, racemicmixtures, and as individual diastereomers, with all possible isomers andmixtures thereof, including optical isomers, being included in thepresent invention. In addition, the cationic lipids disclosed herein mayexist as tautomers and both tautomeric forms are intended to beencompassed by the scope of the invention, even though only onetautomeric structure is depicted.

It is understood that substituents and substitution patterns on thecationic lipids of the instant invention can be selected by one ofordinary skill in the art to provide cationic lipids that are chemicallystable and that can be readily synthesized by techniques known in theart, as well as those methods set forth below, from readily availablestarting materials. If a substituent is itself substituted with morethan one group, it is understood that these multiple groups may be onthe same carbon or on different carbons, so long as a stable structureresults.

It is understood that one or more Si atoms can be incorporated into thecationic lipids of the instant invention by one of ordinary skill in theart to provide cationic lipids that are chemically stable and that canbe readily synthesized by techniques known in the art from readilyavailable starting materials.

In the compounds of Formula A, the atoms may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. The present invention is meant to includeall suitable isotopic variations of the compounds of Formula A. Forexample, different isotopic forms of hydrogen (H) include protium (¹H)and deuterium (²H). Protium is the predominant hydrogen isotope found innature. Enriching for deuterium may afford certain therapeuticadvantages, such as increasing in vivo half-life or reducing dosagerequirements, or may provide a compound useful as a standard forcharacterization of biological samples. Isotopically-enriched compoundswithin Formula A can be prepared without undue experimentation byconventional techniques well known to those skilled in the art or byprocesses analogous to those described in the Scheme and Examples hereinusing appropriate isotopically-enriched reagents and/or intermediates.

As used herein, “alkyl” means a straight chain, cyclic or branchedsaturated aliphatic hydrocarbon having the specified number of carbonatoms.

As used herein, “alkenyl” means a straight chain, cyclic or branchedunsaturated aliphatic hydrocarbon having the specified number of carbonatoms including but not limited to diene, triene and tetraeneunsaturated aliphatic hydrocarbons.

Examples of a cyclic “alkyl” or “alkenyl are:

As used herein, “heterocyclyl” or “heterocycle” means a 4- to10-membered aromatic or nonaromatic heterocycle containing from 1 to 4heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes, thefollowing: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof all of which are optionally substituted with one tothree substituents selected from R″.

As used herein, “polyamine” means compounds having two or more aminogroups. Examples include putrescine, cadaverine, spermidine, andspermine.

As used herein, “halogen” means Br, Cl, F and I.

In an embodiment of Formula A, R¹ and R² are independently selected fromH and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted withone to three substituents selected from R′, or R¹ and R² can be takentogether with the nitrogen to which they are attached to form amonocyclic heterocycle with 4-7 members optionally containing, inaddition to the nitrogen, one or two additional heteroatoms selectedfrom N, O and S, said monocyclic heterocycle is optionally substitutedwith one to three substituents selected from R′.

In an embodiment of Formula A, R¹ and R² are independently selected fromH, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl areoptionally substituted with one to three substituents selected from R′,or R¹ and R² can be taken together with the nitrogen to which they areattached to form a monocyclic heterocycle with 4-7 members optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic heterocycle isoptionally substituted with one to three substituents selected from R′.

In an embodiment of Formula A, R¹ and R² are independently selected frommethyl, ethyl and propyl.

In an embodiment of Formula A, R¹ and R² are each methyl.

In an embodiment of Formula A, R³ is independently selected from: H andmethyl.

In an embodiment of Formula A, R³ is H.

In an embodiment of Formula A, R′ is R″.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl, wherein said methyl, ethyl and propyl areoptionally substituted with one or more halogen and OH.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl.

In an embodiment of Formula A, n is 0, 1, 2, 3 or 4.

In an embodiment of Formula A, n is 2, 3 or 4.

In an embodiment of Formula A, n is 3.

In an embodiment of Formula A, X is O, NR″, (C═O)O, NR″(C═O), O(C═O)O,NR″(C═O)NR″, O(C═O)NR″, or NR″(C═O)O.

In an embodiment of Formula A, X is (C═O)O.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₄ alkyl andC₄-C₂₄ alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₄ alkyl andC₄-C₂₄ alkenyl.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₄ alkenyl.

In an embodiment of Formula A, L₁ is selected from C₁₂-C₂₄ alkenyl.

In an embodiment of Formula A, L₁ is C₁₉ alkenyl.

In an embodiment of Formula A, L₁ is:

In an embodiment of Formula A, L₁ is:

In an embodiment of Formula A, L₂ is selected from C₃-C₉ alkyl and C₃-C₉alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₅-C₉ alkyl and C₅-C₉alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₇-C₉ alkyl and C₇-C₉alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₃-C₉ alkyl and C₃-C₉alkenyl.

In an embodiment of Formula A, L₂ is selected from C₅-C₉ alkyl and C₅-C₉alkenyl.

In an embodiment of Formula A, L₂ is selected from C₇-C₉ alkyl and C₇-C₉alkenyl.

In an embodiment of Formula A, L₂ is C₃-C₉ alkyl.

In an embodiment of Formula A, L₂ is C₅-C₉ alkyl.

In an embodiment of Formula A, L₂ is C₇-C₉ alkyl.

In an embodiment of Formula A, L₂ is C₉ alkyl.

In an embodiment of Formula A, “heterocyclyl” is pyrolidine, piperidine,morpholine, imidazole or piperazine.

In an embodiment of Formula A, “monocyclic heterocyclyl” is pyrolidine,piperidine, morpholine, imidazole or piperazine.

In an embodiment of Formula A, “polyamine” is putrescine, cadaverine,spermidine or spermine.

In an embodiment, “alkyl” is a straight chain saturated aliphatichydrocarbon having the specified number of carbon atoms.

In an embodiment, “alkenyl” is a straight chain unsaturated aliphatichydrocarbon having the specified number of carbon atoms.

Included in the instant invention is the free form of cationic lipids ofFormula A, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the isolated specific cationic lipidsexemplified herein are the protonated salts of amine cationic lipids.The term “free form” refers to the amine cationic lipids in non-saltform. The encompassed pharmaceutically acceptable salts not only includethe isolated salts exemplified for the specific cationic lipidsdescribed herein, but also all the typical pharmaceutically acceptablesalts of the free form of cationic lipids of Formula A. The free form ofthe specific salt cationic lipids described may be isolated usingtechniques known in the art. For example, the free form may beregenerated by treating the salt with a suitable dilute aqueous basesolution such as dilute aqueous NaOH, potassium carbonate, ammonia andsodium bicarbonate. The free forms may differ from their respective saltforms somewhat in certain physical properties, such as solubility inpolar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

The pharmaceutically acceptable salts of the instant cationic lipids canbe synthesized from the cationic lipids of this invention which containa basic or acidic moiety by conventional chemical methods. Generally,the salts of the basic cationic lipids are prepared either by ionexchange chromatography or by reacting the free base with stoichiometricamounts or with an excess of the desired salt-forming inorganic ororganic acid in a suitable solvent or various combinations of solvents.Similarly, the salts of the acidic compounds are formed by reactionswith the appropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the cationic lipids of thisinvention include the conventional non-toxic salts of the cationiclipids of this invention as formed by reacting a basic instant cationiclipids with an inorganic or organic acid. For example, conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like, as well as salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,trifluoroacetic (TFA) and the like.

When the cationic lipids of the present invention are acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the cationic lipids of the present inventionare potentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof. The reagents utilized in synthesizingthe cationic lipids are either commercially available or are readilyprepared by one of ordinary skill in the art.

Synthesis of the novel cationic lipids is a linear process starting fromlipid acid (i). Coupling to N,O-dimethyl hydroxylamine gives the Weinrebamide ii. Grignard addition generates ketone iii. Reduction of theketone generates alcohol iv. Esterification via EDC coupling givesesters of type v.

(20Z 23Z)-nonacosa-20,23-dien-10-yl 4-(dimethylamino)butanoate (Compound1)

11,14-Eicosadienoic acid, (11Z,14Z)— (50 g, 162 mmol),N,O-Dimethylhydroxylamine hydrochloride (3.16 g, 324 mmol), HOAt (44.1g, 324 mmol), Et₃N (45.2 mL, 324 mmol), and EDC (62.1 g, 324 mmol) weremixed in DCM (810 mL) and stirred overnight at ambient temperature.Reaction was then washed 5×700 mL water, then washed 1×600 mL 1 M NaOH,dried with sodium sulfate, filtered through celite and evaporated toobtain 53.06 g (93%) 11,14-eicosadienamide, N-methoxy-N-methyl-,(11Z,14Z) as a clear golden oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H),3.68 (s, 3H), 3.18 (s, 3H), 2.77 (m, 2H), 2.41 (t, J=7 Hz, 2H), 2.05 (m,4H), 1.63 (m, 2H), 1.40-1.26 (m, 18H), 0.89 (t, J=7 Hz, 3H).

11,14-eieosadienamide, N-methoxy-N-methyl-, (11Z,14Z)-1 (4 g, 11.38mmol) was dissolved in dry THF (50.0 ml) in a 250 mL flask then 1 Mnonylmagnesium bromide (22.76 ml, 22.76 mmol) was added under nitrogenat ambient temperature. After 10 min, the reaction was slowly quenchedwith excess sat. aq NH₄Cl. The reaction was washed into a separatoryfunnel with hexane and water, shaken, the lower aqueous layer discarded,the upper layer dried with sodium sulfate, filtered, and evaporated togive crude ketone as a golden oil. The ketone was carried directly intothe next reaction crude.

Ketone (6.32 g, 15.1 mmol, 1 equiv) was dissolved in EtOH (75 mL) andNaBH₄ (2.86 g, 75 mmol, 5 equiv) added. After 15 min of stirring at rt,the reaction was evaporated to a residue, taken up in hexane (200 mL)and water (200 mL), organic layer separated and dried with sodiumsulfate, filtered, and evaporated to 20,23-nonacosadien-10-ol,(20Z,23Z)— (iv) obtained as a white solid which was used without furtherpurification, ¹H NMR (500 MHz, CDCl₃) δ 5.35 (m, 4H), 3.58 (m, 1H), 2.77(m, 2H), 2.05 (m, 4H), 1.48-1.22 (m, 39H), 0.89 (m, 6H).

4-(dimethylamino)butyric acid hydrochloride (3.03 g, 18.05 mmol, 1.2equiv), 20,23-nonacosadien-10-ol, (20Z,23Z)— (iv) (6.33 g, 15.04 mmol, 1equiv), EDC (3.46 g, 18.05 mmol, 1.2 equiv), DMAP (0.368 g, 3.01 mmol,0.2 equiv), and DIEA (7.88 mL, 45.1 mmol, 3 equiv) were combined in DCM(100 mL) and stirred at ambient temperature for 16 hours. The reactionwas then washed 1×250 mL sat. NaHCO₃, and then the lower organic layerdirectly injected onto a 330 g silica column and purified eluting 0-15%MeOH/DCM over 30 min. Collected butanoic acid, 4-(dimethylamino)-,(11Z,14Z)-1-nonyl-11,14-eicosadien-1-yl ester (1) as a yellow oil. ¹HNMR (500 MHz, CDCl₃) δ 5.35 (m, 4H), 4.87 (m, 1H), 2.77 (m, 2H),2.34-2.26 (m, 4H), 2.22 (s, 6H), 2.05 (m, 4H), 1.79 (m, 2H), 1.51 (m,4H), 1.38-1.22 (m, 34H), 0.88 (m, 6H).

(18Z)-heptacos-18-en-10-yl 4-(dimethylamino)butanoate (Compound 2)

Compound 2 was prepared in a manner analogous to that described forCompound 1 above. HRMS (M+H) calc'd 508.5088. found 508.5091.

Compound 3 is MC3 as described in WO 2010/054401, and WO 2010/144740 A1.

Compound 4 is DLinKC2DMA as described in Nature Biotechnology, 2010, 28,172-176, WO 2010/042877 A1, WO 2010/048536 A2, WO 2010/088537 A2, and WO2009/127060 A1.

LNP Compositions

The following lipid nanoparticle compositions (LNPs) of the instantinvention are useful for the delivery of oligonucleotides, specificallysiRNA and miRNA:

Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;

Cationic Lipid/Cholesterol/PEG-DMG 60/38/2;

Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;

Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;

Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;

Cationic Lipid/Cholesterol/PEG-C-DMA/DSPC 40/48/2/10;

Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and

Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10.

LNP Process Description:

The Lipid Nano-Particles (LNP) are prepared by an impinging jet process.The particles are formed by mixing lipids dissolved in alcohol withsiRNA dissolved in a citrate buffer. The mixing ratio of lipids to siRNAare targeted at 45-55% lipid and 65-45% siRNA. The lipid solutioncontains a novel cationic lipid of the instant invention, a helper lipid(cholesterol), PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at aconcentration of 5-15 mg/mL with a target of 9-12 mg/mL in an alcohol(for example ethanol). The ratio of the lipids has a mole percent rangeof 25-98 for the cationic lipid with a target of 35-65, the helper lipidhas a mole percent range from 0-75 with a target of 30-50, the PEG lipidhas a mole percent range from 1-15 with a target of 1-6, and the DSPChas a mole percent range of 0-15 with a target of 0-12. The siRNAsolution contains one or more siRNA sequences at a concentration rangefrom 0.3 to 1.0 mg/mL with a target of 0.3-0.9 mg/mL in a sodium citratebuffered salt solution with pH in the range of 3.5-5. The two liquidsare heated to a temperature in the range of 15-40° C., targeting 30-40°C., and then mixed in an impinging jet mixer instantly forming the LNP.The teeID has a range from 0.25 to 1.0 mm and a total flow rate from10-600 mL/min. The combination of flow rate and tubing ID has effect ofcontrolling the particle size of the LNPs between 30 and 200 nm. Thesolution is then mixed with a buffered solution at a higher pH with amixing ratio in the range of 1:1 to 1:3 vol:vol but targeting 1:2vol:vol. This buffered solution is at a temperature in the range of15-40° C., targeting 30-40° C. The mixed LNPs are held from 30 minutesto 2 hrs prior to an anion exchange filtration step. The temperatureduring incubating is in the range of 15-40° C., targeting 30-40° C.After incubating the solution is filtered through a 0.8 um filtercontaining an anion exchange separation step. This process uses tubingIDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000mL/min. The LNPs are concentrated and diafiltered via an ultrafiltrationprocess where the alcohol is removed and the citrate buffer is exchangedfor the final buffer solution such as phosphate buffered saline. Theultrafiltration process uses a tangential flow filtration format (TFF).This process uses a membrane nominal molecular weight cutoff range from30-500 KD. The membrane format can be hollow fiber or flat sheetcassette. The TFF processes with the proper molecular weight cutoffretains the LNP in the retentate and the filtrate or permeate containsthe alcohol; citrate buffer; final buffer wastes. The TFF process is amultiple step process with an initial concentration to a siRNAconcentration of 1-3 mg/mL. Following concentration, the LNPs solutionis diafiltered against the final buffer for 10-20 volumes to remove thealcohol and perform buffer exchange. The material is then concentratedan additional 1-3 fold. The final steps of the LNP process are tosterile filter the concentrated LNP solution and vial the product.

Analytical Procedure:

1) siRNA Concentration

The siRNA duplex concentrations are determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(RDVs), are treated with 0.5% Triton X-100 to free total siRNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase is composed of A: 25 mMNaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mM Tris,20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1ml/min. The siRNA amount is determined by comparing to the siRNAstandard curve.

2) Encapsulation Rate

Fluorescence reagent SYBR Gold is employed for RNA quantitation tomonitor the encapsulation rate of RDVs. RDVs with or without TritonX-100 are used to determine the free siRNA and total siRNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif.). Samples are excited at 485nm and fluorescence emission was measured at 530 nm. The siRNA amount isdetermined by comparing to the siRNA standard curve.Encapsulation rate=(1−free siRNA/total siRNA)×100%3) Particle Size and Polydispersity

RDVs containing 1 μg siRNA are diluted to a final volume of 3 ml with 1×PBS. The particle size and polydispersity of the samples is measured bya dynamic light scattering method using ZetaPALS instrument (BrookhavenInstruments Corporation, Holtsville, N.Y.). The scattered intensity ismeasured with He—Ne laser at 25° C. with a scattering angle of 90°.

4) Zeta Potential Analysis

RDVs containing 1 μg siRNA are diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples isdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit is assumed in the calculation of zetapotentials,

5) Lipid Analysis

Individual lipid concentrations are determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, Mass.).Individual lipids in RDVs are analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase is composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient changes from 60% mobile phase A and 40% mobile phase B fromtime 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40%mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobilephase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phaseB from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60%mobile phase A and 40% mobile phase B at 20.00 min with flow rate of 1ml/min. The individual lipid concentration is determined by comparing tothe standard curve with all the lipid components in the RDVs with aquadratic curve fit. The molar percentage of each lipid is calculatedbased on its molecular weight.

Utilizing the above described LNP process, specific LNPs with thefollowing ratios were identified:

Nominal Composition:

Cationic Lipid/Cholesterol/PEG-DMG 60/38/2; and

Cationic Lipid/Cholesterol/PEG-DMG DSPC 58/30/2/10.

Oligonucleotide synthesis is well known in the art. (See US patentapplications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US2009/0263407 and US 2009/0285881 and PCT patent applications: WO2009/086558, WO2009/127060, WO2009/132131, WO2010/042877, WO2010/054384,WO2010/054401, WO2010/054405 and WO2010/054406). The Luc and ApoB siRNAincorporated in the LNPs disclosed and utilized in the Examples weresynthesized via standard solid phase procedures.

Luc siRNA

(SEQ. ID. NO.: 1) 5′-iB-A U AAGG CU A U GAAGAGA U ATT-iB 3′(SEQ. ID. NO.: 2) 3′-UUUAUUCCGAUACUUCUC UAU-5′

-   -   AUGC—Ribose    -   iB—Inverted deoxy abasic    -   UC—2′ Fluoro    -   AGT—2′ Deoxy    -   AGU—2′ OCH₃        Nominal Composition        Cationic Lipid/Cholesterol/PEG-DMG 60/38/2        Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10        Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10        ApoB siRNA

(SEQ ID NO.: 3) 5′-iB-CUUUAACAAUUCCUGAAAUTsT-iB-3′ (SEQ ID NO.: 4)3′-UsUGAAAUUGUUAAGGACUsUsUsA-5′

-   -   AUGC—Ribose    -   iB—Inverted deoxy abasic    -   UC—2′ Fluoro    -   AGT—2′ Deoxy    -   AGU—2′ OCH₃    -   UsA—phophorothioate linkage

Example 1 Mouse In Vivo Evaluation of Efficacy

LNPs utilizing Compound 1, in the nominal compositions describedimmediately above, were evaluated for in vivo efficacy. The siRNAtargets the mRNA transcript for the firefly (Photinus pyralis)luciferase gene (Accession 4 M15077). The primary sequence and chemicalmodification pattern of the luciferase siRNA is displayed above. The invivo luciferase model employs a transgenic mouse in which the fireflyluciferase coding sequence is present in all cells.ROSA26-LoxP-Stop-LoxP-Luc (LSL-Luc) transgenic mice licensed from theDana Farber Cancer Institute are induced to express the Luciferase geneby first removing the LSL sequence with a recombinant Ad-Cre virus(Vector Biolabs). Due to the organo-tropic nature of the virus,expression is limited to the liver when delivered via tail veininjection. Luciferase expression levels in liver are quantitated bymeasuring light output, using an IVIS imager (Xenogen) followingadministration of the luciferin substrate (Caliper Life Sciences).Pre-dose luminescence levels are measured prior to administration of theRDVs. Luciferin in PBS (15 mg/mL) is intraperitoneally (IP) injected ina volume of 150 μL. After a four minute incubation period mice areanesthetized with isoflurane and placed in the IVIS imager. The RDVs(containing siRNA) in PBS vehicle were tail vein injected n a volume of0.2 mL. Final dose levels ranged from 0.1 to 0.5 mg/kg siRNA. PBSvehicle alone was dosed as a control. Mice were imaged 48 hours postdose using the method described above. Changes in luciferin light outputdirectly correlate with luciferase mRNA levels and represent an indirectmeasure of luciferase siRNA activity. In vivo efficacy results areexpressed as % inhibition of luminescence relative to pre-doseluminescence levels. Systemic administration of the luciferase siRNARDVs decreased luciferase expression in a dose dependant manner. Greaterefficacy was observed in mice dosed with Compound 1 containing RDVs thanwith the RDV containing the octyl-CLinDMA (OCD) cationic lipid (FIG. 1).OCD is known and described in WO2010/021865.

Example 2 Rat In Vivo Evaluation of Efficacy and Toxicity

LNPs utilizing compounds in the nominal compositions described above,were evaluated for in vivo efficacy and increases in alanine aminotransferase and aspartate amino transferase in Sprague-Dawley(Crl:CD(SD) female rats (Charles River Labs). The siRNA targets the mRNAtranscript for the ApoB gene (Accession # NM 019287). The primarysequence and chemical modification pattern of the ApoB siRNA isdisplayed above. The RDVs (containing siRNA) in PBS vehicle were tailvein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately3 ml/min. Five rats were used in each dosing group. After LNPadministration, rats are placed in cages with normal diet and waterpresent. Six hours post dose, food is removed from the cages. Animalnecropsy is performed 24 hours after LNP dosing. Rats are anesthetizedunder isoflurane for 5 minutes, then maintained under anesthesia byplacing them in nose cones continuing the delivery of isoflurane untilex-sanguination is completed. Blood is collected from the vena cavausing a 23 gauge butterfly venipuncture set and aliquoted to serumseparator vacutainers for serum chemistry analysis. Punches of theexcised caudate liver lobe are taken and placed in RNALater (Ambion) formRNA analysis. Preserved liver tissue was homogenized and total RNAisolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNAisolation kit following the manufacturer's instructions. Liver ApoB mRNAlevels were determined by quantitative RT-PCR. Message was amplifiedfrom purified RNA utilizing a rat ApoB commercial probe set (AppliedBiosystems Cat # RN01499054_m1). The PCR reaction was performed on anABI 7500 instrument with a 96-well Fast Block. The ApoB mRNA level isnormalized to the housekeeping PPIB (NM 011149) mRNA. PPIB mRNA levelswere determined by RT-PCR using a commercial probe set (AppliedBiosytems Cat. No. Mm00478295_m1). Results are expressed as a ratio ofApoB mRNA/PPIB mRNA. All mRNA data is expressed relative to the PBScontrol dose. Serum ALT and AST analysis were performed on the SiemensAdvia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanineaminotransferase (Cat#03039631) and aspartate aminotransferase(Cat#03039631) reagents. Similar efficacy was observed in rats dosedwith Compound 2 containing RDV than with the RDV containing the cationiclipid DLinKC2DMA (Compound 4) or MC3 (Compound 3, FIG. 2). Additionally,3 out of 4 rats treated with 3 mg/kg DLinKC2DMA (Compound 4) failed tosurvive 48 hours and 2 out of 4 rats treated with 3 mg/kg MC3 (Compound3) failed to survive 48 hours. 3 out of 4 rats treated with 6 mg/kgCompound 2 survived at 48 hours post dose.

Example 3 Determination of Cationic Lipid Levels in Rat Liver

Liver tissue was weighed into 20-ml vials and homogenized in 9 v/w ofwater using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 μL aliquot of each tissue homogenate was mixed with 300 μL ofextraction/protein precipitating solvent (50/50 acetonitrile/methanolcontaining 500 nM internal standard) and the plate was centrifuged tosediment precipitated protein. A volume of 200 μL of each supernatantwas then transferred to separate wells of a 96-well plate and 10 μlsamples were directly analyzed by LC/MS-MS.

Standards were prepared by spiking known amounts of a methanol stocksolution of compound into untreated rat liver homogenate (9 volwater/weight liver). Aliquots (50 μL) each standard/liver homogenate wasmixed with 300 μL of extraction/protein precipitating solvent (50/50acetonitrile/methanol containing 500 nM internal standard) and the platewas centrifuged to sediment precipitated protein. A volume of 200 μL ofeach supernatant was transferred to separate wells of a 96-well plateand 10 μl of each standard was directly analyzed by LC/MS-MS.

Absolute quantification versus standards prepared and extracted fromliver homogenate was performed using an Aria LX-2 HPLC system (ThermoScientific) coupled to an API 4000 triple quadrupole mass spectrometer(Applied Biosystems). For each run, a total of 10 μL sample was injectedonto a BDS Hypersil C8 HPLC column (Thermo, 50×2 mm, 3 μm) at ambienttemperature.

Mobile Phase A:

95% H2O/5% methanol/10 mM ammonium formate/0.1% formic acid Mobile PhaseB: 40% methanol/60% n-propanol/10 mM ammonium formate/0.1% formic acidThe flow rate was 0.5 mL/min and gradient elution profile was asfollows: hold at 80% A for 0.25 mM, linear ramp to 100% B over 1.6 min,hold at 100% B for 2.5 min, then return and hold at 80% A for 1.75 min.Total run time was 5.8 min. API 4000 source parameters were CAD: 4, CUR:15, GS1: 65, GS2: 35, IS: 4000, TEM: 550, CXP: 15, DP: 60, EP: 10. Inrats dosed with Compound 2 containing RDV liver levels were lower thanwith the RDV containing the cationic lipid DLinKC2DMA (Compound 4) orMC3 (Compound 3, FIG. 3).

What is claimed is:
 1. A lipid nanoparticle comprising anoligonucleotide and a lipid of Formula A:

wherein: R¹ and R² are independently selected from H, (C₁-C₆)alkyl,heterocyclyl, and polyamine, wherein said alkyl, heterocyclyl andpolyamine are optionally substituted with one to three substituentsselected from R′, or R¹ and R² can be taken together with the nitrogento which they are attached to form a monocyclic heterocycle with 4-7members optionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclicheterocycle is optionally substituted with one to three substituentsselected from R′; R³ is selected from H and (C₁-C₆)alkyl, said alkyloptionally substituted with one to three substituents selected from R′;R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ orCON(R″)₂; R″ is independently selected from H and (C₁-C₆)alkyl, alkyl isoptionally substituted with halogen and OH; n is 0, 1, 2, 3, 4 or 5; Xis selected from O, NR″, (C═O)O, NR″(C═O), O(C═O)O, NR″(C═O)NR″,O(C═O)NR″, and NR″(C═O)O; L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄alkenyl, said alkyl and alkenyl are optionally substituted with one ormore substituents selected from R′; and L₂ is selected fromunsubstituted C₉ alkyl and unsubstituted C₉ alkenyl; or anypharmaceutically acceptable salt or stereoisomer thereof.
 2. The lipidnanoparticle of claim 1, wherein in the lipid of Formula A R₁ and R₂ areeach methyl; R³ is H; n is 3; X is (C═O)O; L₁ is selected from C₄-C₂₄alkyl and C₄-C₂₄ alkenyl; and L₂ is selected from C₃-C₉ alkyl and C₃-C₉alkenyl; or any pharmaceutically acceptable salt or stereoisomerthereof.
 3. The lipid nanoparticle of claim 1, wherein the lipid is:(20Z,23Z)-nonacosa-20,23-dien-10-yl 4-(dimethylamino)butanoate (Compound1); and (18Z)-heptacos-18-en-10-yl4-(dimethylamino)butanoate (Compound2); or any pharmaceutically acceptable salt or stereoisomer thereof. 4.The lipid nanoparticle according to claim 1, wherein the oligonucleotideis siRNA or miRNA.
 5. The lipid nanoparticle according to claim 1,wherein the oligonucleotide is siRNA.
 6. The lipid nanoparticle of claim1, wherein L₁ is C₄-C₂₄ alkenyl.
 7. The lipid nanoparticle of claim 6,wherein L₁ is C₁₂-C₂₄ alkenyl.
 8. The lipid nanoparticle of claim 7,wherein L₁ is C₁₉ alkenyl.
 9. The lipid nanoparticle of claim 7, whereinL₁ is


10. The lipid nanoparticle of claim 1, wherein R₁ and R₂ areindependently selected from H, methyl, ethyl and propyl.
 11. The lipidnanoparticle of claim 10, wherein R₁ and R₂ each are methyl.
 12. Thelipid nanoparticle of claim 1, wherein R₃ is H or methyl.
 13. The lipidnanoparticle of claim 12, wherein R₃ is H.
 14. The lipid nanoparticle ofclaim 1, wherein n is 2, 3 or
 4. 15. The lipid nanoparticle of claim 14,wherein n is
 3. 16. The lipid nanoparticle of claim 1, wherein X is O,NR″, (C═O)O, NR″(C═O), O(C═O)O, NR″(C═O)NR″, O(C═O)NR″ or NR″C(C═O)O.17. The lipid nanoparticle of claim 16, wherein X is (C═O)O.